NFY和RFX1对人PNRC启动子的调控作用

Regulation of Human PNRC Promoter Activity by NFY and RFX1

探讨核因子Y(nuclear factor Y,NFY)和调节因子X1(regulatory factors that bind to the X box,RFX1)对人PNRC(proline-rich nuclear receptor coactivator)基因的调控作用及机制.根据凝胶电泳迁移率变化实验,分析NFY和RFX1与PNRC启动子区域的结合.将含有NFY和RFX1的真核表达质粒(pCMV-NFY,pCMV-RFX1)和含有PNRC启动子的荧光素酶报告基因质粒共转染HepG2细胞,检测转染细胞的荧光素酶活性,并用RT-PCR和Western印迹检测PNRC的表达情况.Quick-Change法对PNRC启动子区NFY和RFX1结合位点进行突变,将包含突变点的重组荧光素酶报告质粒与含有NFY和RFX1的真核表达质粒共同转染HepG2细胞,检测各组荧光素酶活性.结果发现,NFY和RFX1能与PNRC启动子区域特异性结合;转染pCMV-NFY和pCMV-RFX1可抑制PNRC启动子活性并下调PNRC在HepG2细胞中的表达;包含NFY和RFX1结合位点的突变质粒与pCMV-NFY和pCMV-RFX1共转染后,NFY和RFX1对PNRC启动子活性的抑制作用消失.以上结果提示,NFY和RFX1能调控PNRC基因的表达,其机制是与PNRC启动子区域的特异性结合位点相结合,发挥其反式抑制作用.

This study was to investigate the regulatory mechanism of the proline-rich nuclear receptor coactivator （PNRC） promoter activity by nuclear factor Y （NFY） and regulatory factors that bind to the X box （RFX1）. The NFY and RFX1 expression vectors （pCMV-NFY and pCMV-RFX1） and the luciferase reporter vector containing the PNRC promoter were co-transfected into HepG2 cells. The luciferase activity of transfected cells was determined and the expression of PNRC was detected by RT-PCR and Western blotting. The bindings of NFY and RFX1 with the PNRC promoter were detected by electrophoretic mobility shift assay （EMSA）. In addition, the binding sites of NFY and RFX1 in the PNRC promoter were mutated by QuikChange method and constructed into luciferase reporter vectors and then co-transfected with pCMV-NFY and pCMV-RFX1 into HepG2 cells for luciferase assays. The EMSA and super-shift results demonstrated the specific binding of NFY and RFX1 proteins to the human PNRC promoter. Transient transfections and luciferase assays further revealed that over-expression of NFY and RFX1 repressed promoter activity of PNRC and down-regulated PNRC expression of HepG2 cells. Whereas, the effects were abolished when PNRC promoter mutant was used in the cotransfection. These results demonstrated that NFY and RFX1 specifically bind to the promoter region of PNRC and repress its activity.